Glyceraldehyde 3 phosphate dehydrogenase gapdh sc 365062

LNCaP and 22Rv1 cells were obtained from the American Type Culture Collection in 2012 (ATCC). C4‐2 cells were obtained from MD Anderson Cancer Center Cell Line Core Facility in 2016 (Houston, TX). All cells were maintained in Rosewell Park Memorial Institute supplemented with 10% fetal bovine serum. Cell line authentication was performed using short tandem repeat profiling (GenePrint 10 kit, Promega). Mycoplasma detection is performed on a plate luminometer using a mycoplasma enzyme‐based luciferase assay (MycoAlert PLUS Mycoplasma Detection Kit, Lonza). Low‐passage (<15) cultures were used for all experimental testing, Enzalutamide (MDV3100), venetoclax (ABT‐199), navitoclax (ABT‐263), A‐1210477, obatoclax, MK2206, and buparlisib were purchased from Selleck Chemicals. Antibodies for Western blot analysis include glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (sc‐365062) and tubulin (sc‐8035): Santa Cruz Biotechnologies; NOXA (114C307): Novus Biologicals; PARP‐1 cleaved (5625), BCL‐2 (4223), BCL‐xL (2764), MCL‐1 (5433), BAX (5023), BIM 2933), BAD (9239), pBAD‐Ser136 (4366), Akt (4691), and pAkt‐Ser473 (4060): Cell Signaling Technology.

Monolayer cells and spheroids were washed with phosphate-buffered saline and lysed in 150 µL of RIPA buffer (50 mM Tris-HCl (pH 7.9), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)). After freezing the lysate at −80°C, the protein concentration was determined by the BCA assay (Nacalai), and was adjusted with the RIPA buffer followed by mixing with 2× Laemmli sample buffer (10% β-mercaptoethanol, 125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.004% bromophenol blue). The samples were subjected to SDS-polyacrylamide gel electrophoresis. Following transfer to the PVDF membranes, immunoblotting was performed. Antibodies against CD44 (#3570; Cell Signaling Technology, Danvers, MA, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-365062; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and p21^CDKN1A (sc-397-G; Santa Cruz Biotechnology) were used.